COMPOSITION CONTAINING  DIHOMO-y-LINOLENIC ACID (DGLA) AS THE ACTIVE INGREDIENT

ABSTRACT

The present invention provides a composition, such as a food and pharmaceutical agent, which comprises dihomo-γ-linolenic acid, and which has the effect of preventing or treating skin diseases; a composition such as a food and pharmaceutical agent which comprises dihomo-γ-linolenic acid and which has the effect of preventing or treating skin diseases; and a composition which comprises dihomo-γ-linolenic acid and which has the effect of preventing or treating diseases related to increased mast cell count.

TECHNICAL FIELD

The present invention relates to a food ingredient having a preventiveor therapeutic effect on various skin diseases including allergicdermatitis, such as atopic dermatitis and contact dermatitis, eczema,and UV-derived skin disorders, or a food composition comprising saidingredient. More specifically, it is intended to provide an optimumdosage for exhibiting the effect of the ingredient to the fullest.

The present invention also relates to a food ingredient having apreventive or therapeutic effect on various diseases such as skindiseases, respiratory system diseases and digestive system diseases thatare closely related to eosinophil infiltration and increased cellcounts, or a food composition or pharmaceutical composition comprisingthe ingredient, and in particular to an inhibitor of eosinophilinfiltration.

Furthermore, the present invention relates to a food ingredient having apreventive or therapeutic effect on various disease conditions such asatopic dermatitis, bronchial asthma, pollinosis, allergic rhinitis andallergic conjunctivitis in which mast cell count are enhancedspecifically in the lesion, or a food composition or a pharmaceuticalcomposition comprising the ingredient, and in particular to an inhibitorof enhancement in mast cell count.

BACKGROUND ART

In the classification of a variety of fatty acids from a structuralviewpoint, those having a long fatty acid chain comprising about 20carbons in the molecule and containing two or more unsaturated sites(double bonds) are termed as polyunsaturated fatty acids (PUFAs).Alternatively, based on its extreme significance for the maintenance ofhuman health from a nutritional viewpoint, some fatty acids are oftenexpressed as essential fatty acids (EFAs). The definition of EFAsignifies, in a narrow sense, linoleic acid (LA) and α-linolenic acid(ALA) that cannot be synthesized by humans and thus must be ingestedthrough food, and in a broad sense, it also includes their metabolites,arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA). Among them, a series of fatty acids produced with ALA as theparent fatty acid are termed as n3-series PUFAs, which include EPA andDHA.

On the other hand, as shown in FIG. 1, similar fatty acids produced withLA as the parent fatty acid are termed as n6-series PUFAs, in which,specifically, LA is metabolized by Δ6-desaturase to γ-linolenic acid(GLA), then by a carbon-chain elongase to DGLA, and further byΔ5-desaturase to AA.

The physiological role of these n6-series PUFAs have been extensivelystudied, and GLA, among them, has been demonstrated to be useful in skindisorders (Cosmetic & Toiletries, Nissen H P, 1995, 119: 71), diabetesmellitus and its complications (Diabetic Medicine, Jamal G A, 1990, 7:319), rheumatoid arthritis (Arthritis Rhermatism, Zurier R B, 1996, 39:1808) and the like. In connection with the molecular mechanism in thephysiological function of GLA, it has been proposed, as shown in FIG. 1,that the active molecule is DGLA which is a carbon chain-elongatedmetabolite of GLA, the 1-series prostaglandin (PG1) which is acyclooxygenase metabolite of DGLA, or 15-hydroxyeicosatrienoic acid(15-HETrE) which is a lipoxygenase metabolite of DGLA.

On the other hand, on the physiological activity of DGLA, part of theeffect has been confirmed in vivo or in vitro: in vivo, effects ofinhibiting platelet agglutination (British Medical Journal, Kernoff P BA, 1977, 2: 1441), delayed-type footpad edema (Lipids, Taki H., 1993,28: 873), blood pressure increase (Lipids, Cedric H., 1984, 19: 699) andother effects have been reported, and in vitro, effects of inhibitingthe production of cytokines such as interleukin-2, -10 and tumornecrosis factor (TNF-α) (Immunology, Maaike M B W D, 2003, 110: 348; TheJournal of Immunology, Deniela S, 1989, 143: 1303), leukotrieneproduction (Archives of Dermatological Research, Iversen L, 1992, 284:222), and T cell growth (Prostaglandin Leukotrienes and Essential FattyAcids, Zurier R B, 1999, 60: 371) and other effects have been reported.

The relationship of the physiological functions of living organisms (inparticular, the skin) and PUFAs have been investigated in variousfields. For example, it has been demonstrated that when animals such asrats are kept under an EFA-deficient condition for a long period oftime, they may develop skin scale, decreased moisture, increasedmoisture loss and the like, and specifically abnormal skin functions. Itis also suggested that PUFAs are deeply involved in various skindiseases such as eczema, contact dermatitis and UV-derived skin damages.Furthermore, GLA is useful in the prevention and treatment of variousdiseases, as described above, and among them, it has been proved, GLA isuseful in skin diseases, specifically in atopic dermatitis (AmericanJournal of Clinical Nutrition, Harrobin D F, 2000, 71: 367).

At first, it was found that in the serum of patients with atopicdermatitis, the amount of LA in the serum is increased as compared tonormal healthy subjects, and conversely, at that time, the amounts ofDGLA and AA that are metabolites from LA were found to be decreased.This fact strongly suggests the possibility that the function ofΔ6-desaturase, an enzyme that converts LA to GLA, in the n6-series PUFAmetabolic pathway is decreased in patients with atopic dermatitis.

Thus, it can be estimated that the ingestion of PUFAs which are locateddownstream to the Δ6-desaturase can improve diseases accompanied byabnormal PUFA metabolism, and it was investigated whether the ingestionof GLA, among them, which is relatively abundant in nature and abundantin the seeds of Oenothera, Ribes nigrum and Borrago officinalis canimprove various conditions of atopic dermatitis.

As a result, it was reported in a human study that the oral ingestion ofGLA (about 180-1440 mg/person/day) can improve the skin inflammationconditions or itching sensations of atopic dermatitis, and specificallyit was more effective in patients who took a high dose of 720 or 1440mg/person/day. The finding at the time confirmed that GLA caneffectively increase the amounts of DGLA and AA in the living body, moreeffectively the amount of DGLA, and that there is a positive correlationbetween the improvement in disease conditions and DGLA (ProstaglandinsLeukotrienes and Medicine, Mauku M S, 1982, 9: 615; The Lancet, WrightS, 1982, 20: 1120).

The effectiveness of GLA has also been confirmed in NC/Nga mice, ananimal model of atopic dermatitis, and the oral ingestion of GLA (about1250 mg/kg/day) has also been found to have effects of inhibiting theformation of atopic dermatitis and IgE production (Abstract of the 50thGeneral Meeting of the Japanese Society of Allergology, Zui Hamada,2000, pp. 999). As described above, the ingestion of GLA, among then6-series PUFAs, is effective for the improvement of atopic dermatitis,and it has been estimated that preferably the ingestion of DGLA that isbelieved to be the active substance is more effective.

However, there remains a possibility that the appropriate establishmentof an optimum dosage has not been made in GLA ingestion intended for thecorrection of abnormal PUFA metabolism such as atopic dermatitismentioned above. In guinea pigs, a phenomenon was observed that thedosage and the amount increased of PUFAs in the living body does notcorrelate, i.e. in the administration of GLA at a highly excessiveamount of about 3200 mg/kg/day, the amount of DGLA in the epidermisdecreased as compared to that when about 400 mg/kg/day of GLA wasingested (Prostaglandins Leukotrienes and Essential Fatty Acids,Navarette R, 1992, 46: 139). This suggests a possibility that theingestion of a large amount of GLA may inhibit the conversion of GLA toDGLA.

Thus, when a large amount of GLA is ingested, the amount of DGLA in theliving body may tend to decrease rather than to increase, posing a riskof aggravating atopic dermatitis. Furthermore, it is known that thereexists an individual difference in the metabolism of GLA in humans. WhenGLA was administered to human patients with atopic dermatitis, thedegree of enhancement in the amount of DGLA in the erythrocyte membranevaried with individuals, and in the patient group having enhancement inthe amount of DGLA the dermatitis condition improved, whereas noimprovement was noted in the patient group having no enhancement in theamount of DGLA (British Journal of Dermatology, Henz B M, 1999, 140:685). This means that the ingestion of GLA is not necessarily effectivefor increasing the amount of DGLA, and that in the treatment of atopicdermatitis, the optimum amount of GLA may not be the optimum amount ofDGLA.

On the other hand, it has been confirmed that the ingestion of DGLAleads to increases in the amount of DGLA in the living body in a dosedependent manner (Abstract of the 58th Meeting of the Japanese Societyof Nutrition and Food Science, Chika Horikawa, 2004, pp. 219), and thuseven if DGLA was ingested in a large amount, it is hard to conceive thatthe amount of DGLA in a living body decreases. Furthermore, in theinhibition of metabolic enzymes by GLA described above, originally theactivity of the enzyme is considered to be relatively high, whichindicates a possibility that carbon-chain elongation enzymes possiblyfree of activity reduction due to aging may be affected by the substrateenvironment or other factors as long as they participate in enzymereactions.

As a result, it is likely that the amount of DGLA when GLA was ingestedmay vary depending on various conditions, which strongly suggests thedifficulty of appropriately controlling the treatment of atopicdermatitis by the ingestion of GLA. Thus, from the viewpoint of safetyand effectiveness, the direct ingestion of DGLA per se is more preferredthan the ingestion of GLA in the treatment or prevention of atopicdermatitis, in which it is further considered to be very important toprovide an optimum dosage.

However, some meat, eggs and seafoods contain DGLA, but they are limitedin the number of types, and for vegetarians, ingestion of DGLA fromnatural products is very difficult. Furthermore, DGLA occurs in nature,but the amount is very limited and mass production is difficult, andthus it is very difficult to demonstrate its effect on atopic dermatitisusing DGLA as in the GLA study mentioned above. However, as far as weknow, there is no direct demonstration that atopic dermatitis isimproved by DGLA per se, and though DGLA has been demonstrated to havevarious physiological effects in in vivo tests or in vitro tests usingvarious animal- or human-derived cell lines, as described above, none ofthe tests are considered to simulate atopic dermatitis, and thus thereis no definite answer to whether DGLA can improve atopic dermatitis ornot.

As shown in U.S. Pat. No. 3,354,581, by inventing a method of obtainingmicroorganisms deficient in Δ5-desaturase and producing DGLA lipids byfermentation of the microorganisms, the present inventors have enabledmass production of a triglyceride SUNTGD of which about 40% ofconstituent fatty acids comprises DGLA, and thus have overcome theprevious difficulties in obtaining the raw material of DGLA.

In recent years, it has been demonstrated that eosinophil infiltrationoccurs at inflammatory regions in various diseases including skindiseases such as atopic dermatitis, eczema and psoriasis, respiratorysystem diseases such as bronchial asthma, chronic obstructive pulmonarydisease (COPD), hypersensitivity pneumonitis and eosinophilicpneumonitis, and digestive system diseases such as eosinophilicgastroenteritis and ulcerative colitis, and thus eosinophils have beenimplicated to be deeply involved in the formation and progression ofthese disease conditions. Under these circumstances, attempts have beenmade to prevent and/or treat these diseases by inhibiting the steps ofeosinophil infiltration into the tissues of the lesion, specificallyeosinophil activation, adhesion to the endothelium, extravascularmigration, and movement of chemotactic factors into the lesion (AmericanJournal of Clinical Dermatology, Chari S, 2001, 2: 1; PaediatricRespiratory Reviews, McMillan R M, 2001, 2: 238; Agents Actions,Rask-Madsen J, 1992, C37; Japanese Unexamined Patent Publication (Kokai)No. 8-3036).

For example, it has been found that steroid external preparations andimmunosuppressive external preparations for which clinical usefulnesshas been confirmed for the treatment of skin diseases can alleviate theaggravation of skin conditions in an atopic dermatitis animal model,NC/Nga mice, and also to suppress significantly the number ofeosinophils infiltrating into the skin lesions (Japanese Journal ofPharmacology, Hiroi J, 1998, 76: 175). Also, as a substance that is achemotactic factor having a property of accumulating eosinophils intothe lesions, leukotriene B4 can be mentioned, and this substance hasbeen found to be synthesized by 5-lipoxygenase.

In contrast, 5-lipoxygenase inhibitors suppress the production ofleukotriene B4 by inhibiting the activity of this enzyme, and alsosuppress the subsequent biological event of eosinophil infiltration. Asa result, it has been found, the compounds can alleviate respiratorydisorders of nocturnal asthma in which leukotrienes and eosinophils aredeeply involved, indicating their usefulness in respiratory systemdiseases (American Journal of Respiratory and Critical Care Medicine,Wenzel S E, 1995, 152: 897). Thus, these pharmaceutical drugs have beendemonstrated to be useful in the prevention and/or treatment of variousdiseases by controlling abnormal eosinophil conditions, whereas thereare problems with safety and the methods for using them.

There are side effects in using steroid external preparations, such asblushing and atrophy of the skin during use and the rebound phenomenonin which the suspension of administration may aggravate dermatitis,whereas immunosuppressive external preparations may facilitate skintumors and their efficacy is greatly affected by the application siteand the state of the barrier function of the skin (Journal of theJapanese Dermatological Association, Masutaka Furue, 2004, 114: 135),and 5-lipoxygenase inhibitors must be orally administered as many asfour times a day and their ingestion over a long period of time maycause dyspepsia (ZYFLO™ FLIMTAB™, package insert, Abbott Laboratories,1998).

Under these circumstances, materials have long been sought that aremedically useful, which can be used safely by everyone, and caneffectively inhibit the function of eosinophils.

Considering medically or nutritionally useful food ingredients, therecan be mentioned eicosapentaenoic acid (EPA) or docosahexaenoic acid(DHA) that are n3-series polyunsaturated fatty acids as shown in FIG. 2as one of the candidate materials. These ingredients are relativelyabundant in marine animal oils, specifically fish oils such as bonitoand sardine oils, which have been ingested by mankind for a long periodof time and are naturally very safe. Also, extensive studies have beenmade on the physiological functions thereof, and such usefulness hasbeen found as the effect of inhibiting blood clots for the former and asthe effect of enhancing the learning function has been demonstrated forthe latter (New Developments in Functional Lipids (Kinousei ShishitsunoShintennkai), Osamu Suzuki, 2001).

As another useful physiological effect, there can be mentioned ananti-allergy effect (The European Respiratory Journal, Nagatsuka T.2000, 16: 861; The Journal of Infectious Diseases, McMurray D N, 2000,182: 861), and one of the mechanisms thereof is proposed to be an effecton the function of immunocompetent cells. As one of the studies thatsupport this, as shown in Japanese Unexamined Patent Publication (Kokai)No. 10-1434 to Yazawa et al., the effect of EPA and DHA on eosinophilmigration has been investigated, and revealed that the intraperitonealadministration of 100 mg of EPA ethylester or 50 mg of DHA ethylestercan inhibit eosinophil migration in the delayed type allergy in guineapigs.

However, this experiment is a confirmation of a biological reaction whenEPA and DHA were intraperitoneally administered, and cannot beconsidered to be an experiment that simulates a situation in which theywere actually ingested as a foodstuff. When intraperitonealadministration and oral administration are compared, it can be easilyconjectured that the concentrations of EPA and DHA are overwhelminghigher in the abdominal cavity, in which eosinophils can infiltrate, inthe former, i.e. the physiological activity can be more easily detected.Thus, it is unknown whether oral ingestion provides such effects or not.

There are also problems of parts being easily oxidized and offensiveodors resulting from the structural characteristics of EPA and DHA. EPAand DHA have unsaturated bonds in their molecules, or parts that areeasily oxidized: five in EPA and six in DHA. By undergoing oxidation,there is quality deterioration, and the possibility cannot be ruled outthat not only does it impair the physiological functions describedabove, but the oxidation products formed may be detrimental to a livingbody. Furthermore, these oxidation products of PUFAs are known to emitexceptionally bad orders, and become worse with accelerated oxidationover time.

As a countermeasure against easily oxidized EPA and DHA, antioxidants,deodorants, masking agents, etc., have been contrived. However, becauseof various problems in that their efficacy for preventing oxidation andthat its duration are not satisfactory and some additives may cloud theappearance, none are considered effective preventive measures (JapaneseUnexamined Patent Publication (Kokai) No. 2-55785, Japanese UnexaminedPatent Publication (Kokai) No. 3-100093, Japanese Unexamined PatentPublication (Kokai) No. 2004-137420). Despite the useful biologicalactivity of EPA and DHA, it is difficult to stably maintain theirquality even with a variety of measures, which represents one of thereasons that limit the range of application into foodstuffs.

From the foregoing, food materials that are medically useful and thatare excellent in safety and quality stability are being sought after.

In recent years, because of changes in the environment and eatinghabits, genetic factors and the like, the number of patients afflictedwith allergic diseases has increased. Pathological conditions ofallergic diseases are roughly divided into type I to type IV based onthe mechanism of pathogenesis and the related functional molecules. Mastcells are considered to be closely related to type a I allergy, alsotermed as delayed type allergy, among them. In a type I allergy, when aliving body is exposed to a certain antigen, an antigen-specific IgEantibody is produced by B cells via antigen presenting cells and helperT cells.

Subsequently, IgE antibody binds to the surface of mast cells to enterinto the state of a guard against the reentry of antigen. Mast cells inthis state capture antigen that enter the surface of the membrane, andinitiate degranulation so as to release various chemical mediators suchas histamine and leukotrienes. The subsequent binding of these chemicalmediators to the receptors causes so-called “allergic reactions” i.e.physiological phenomena that are detrimental to humans such as edema,reddening, itching, airway resistance and enhanced mucous secretion.

Under these circumstances, various attempts for alleviating allergicreactions have been made, by inhibiting the function of mast cells,specifically by a method of suppressing the degranulation of mast cellsto suppress the release of chemical mediators, a method of suppressingthe synthesis of chemical mediators, or a method of inhibiting thebinding of the released chemical mediators and receptors, specificallyby suppressing the actions of chemical mediators produced by mast cells.It is recognized now that many of the pharmaceutical agents used foralleviating itching associated with atopic dermatitis, and treatingpollinosis, allergic conjunctivits etc., are based on any of themechanisms described above (Internet HP “The Rheumatism & AllergyInformation Center”, Maki Hasegawa, 2005 Apr. 4, Allergic inflammatorydiseases—New approaches, Motohiro Kurosawa, 1994).

However, some reports describe that not only the function of the abovechemical mediators produced by mast cells, but the number per se of mastcells identified in the lesion are changed. For example, it has beenfound that the differentiation and proliferation of mast cells in thenasal mucosa of patients with allergic rhinitis are more frequent thanthose with nonallergic diseases (Journal of the Oto-Rhino-LaryngologicalSociety of Japan (Nippon Jibiinkoka Gakkai Kaiho), Naoshi Yoshida, 2001,104: 504). It has also been revealed that patients who have asthma, thenumber of mast cells localized in bronchial smooth muscles aresignificantly greater than normal healthy subjects (Current Opinion inAllergy and Clinical Immunology, Peter B, 2003, 3: 45).

There are similar reports on skin diseases, for example increases inmast cells have been reported in lesions, etc., of basal cell carcinoma,psoriasis vulgaris and atopic dermatitis (Allergy, Shoso Yamamoto, 2000,49: 455). In other words, as a means for suppressing and/or alleviatingdiseases closely related to enhanced mast cell count, it is importantnot only to control the amount and function of chemical mediatorsproduced by inflammatory cells, specifically mast cells, but also tosuppress the abnormal growth of the mast cells per se to keep the numberof these cells at normal levels, and it is believed that by controllingthe processes, more effective and multi-faceted prevention and treatmentof diseases can be attained.

For example, it has been found that steroid external preparations,immunosuppressive external preparations or the like of which clinicalsignificance has been recognized against allergic dermatitis, etc., notonly exhibit significant improvement in the dermatitis symptom scoreassociated with dermatitis, but also significantly suppress increases inthe number of mast cells in the lesions of dermatitis in the NC/Nga micewhich is an animal model of allergic dermatitis (Japanese Journal ofPharmacology, Hiroi J, 1998, 76: 175). This also strongly suggests thatthe therapeutic effect and mast cell count are correlated, and supportsthe importance of regulating mast cell count at an appropriate level.

Thus, these pharmaceutical drugs have been demonstrated to be useful inthe prevention and/or treatment of various mast cell-related diseasessuch as allergic diseases, whereas they have problems in safety andmethods of use. Steroid external preparations have side effects, such asblushing, atrophy of the skin, and a rebound phenomenon in whichsuspending administration may aggravate dermatitis, whereasimmunosuppressive external preparations have problems in that their usemay facilitate skin tumors and their efficacy is greatly affected by theapplication site and the state of the barrier function of the skin(Journal of the Japanese Dermatological Association, Masutaka Furue,2004, 114: 135).

Under these circumstances, materials have long been sought that aremedically useful, that can be used safely by everyone, and that caneffectively suppress enhancement in the number of mast cells.

-   Patent document 1: U.S. Pat. No. 3,354,581-   Patent document 2: Japanese Unexamined Patent Publication (Kokai)    No. 8-3036-   Patent document 3: Japanese Unexamined Patent Publication (Kokai)    No. 2-55785-   Patent document 4: Japanese Unexamined Patent Publication (Kokai)    No. 3-100093-   Patent document 5: Japanese Unexamined Patent Publication (Kokai)    No. 2004-137420-   Nonpatent document 1: Cosmetic & Toiletries, Nissen H P, 1995, 119:    71-   Nonpatent document 2: Diabetic Medicine, Jamal G A, 1990, 7: 319-   Nonpatent document 3: Arthritis Rhermatism, Zurier R B, 1996, 39:    1808-   Nonpatent document 4: British Medical Journal, Kernoff P B A, 1977,    2: 1441-   Nonpatent document 5: Lipids, Taki H., 1993, 28: 873-   Nonpatent document 6: Lipids, Cedric H., 1984, 19: 699-   Nonpatent document 7: Immunology, Maaike M B W D, 2003, 110: 348-   Nonpatent document 8: The Journal of Immunology, Deniela S, 1989,    143: 1303-   Nonpatent document 9: Archives of Dermatological Research, Iversen    L, 1992, 284: 222-   Nonpatent document 10: Prostaglandin Leukotrienes and Essential    Fatty Acids, Zurier R B, 1999, 60: 371-   Nonpatent document 11: American Journal of Clinical Nutrition,    Harrobin D F, 2000, 71: 367-   Nonpatent document 12: Prostaglandins Leukotrienes and Medicine,    Mauku M S, 1982, 9: 615-   Nonpatent document 13: The Lancet, Wright S, 1982, 20: 1120-   Nonpatent document 14: Abstract of the 50th General Meeting of the    Japanese Society of Allergology, Zui Hamada, 2000, pp. 999-   Nonpatent document 15: Prostaglandins Leukotrienes and Essential    Fatty Acids, Navarette R, 1992, 46: 139-   Nonpatent document 16: British Journal of Dermatology, Henz B M,    1999, 140: 685-   Nonpatent document 17: Abstract of the 58th meeting of the Japanese    Society of Nutrition and Food Science, Chika Horikawa, 2004, pp. 219-   Nonpatent document 18: New Developments in Functional Lipids    (Kinosei Shishitsuno Shintennkai), Osamu Suzuki, 2001-   Nonpatent document 19: γ-Linolenic Acid, Recent Advances in    Biotechnology and Clinical Applications, Hundy Y S, 2001-   Nonpatent document 20: American Journal of Clinical Dermatology,    Chari S, 2001, 2: 1-   Nonpatent document 21: Paediatric Respiratory Reviews, McMillan R M,    2001, 2: 238-   Nonpatent document 22: Agents Actions, Rask-Madsen J, 1992, C37    (Japanese Unexamined Patent Publication (Kokai) No. 8-3036)-   Nonpatent document 23: Japanese Journal of Pharmacology, Hiroi J,    1998, 76: 175-   Nonpatent document 24: American Journal of Respiratory and Critical    Care Medicine, Wenzel S E, 1995, 152: 897-   Nonpatent document 25: Journal of the Japanese Dermatological    Association, Masutaka Furue, 2004, 114: 135-   Nonpatent document 26: ZYFLO™ FLIMTAB, the package insert, Abbott    Laboratories, 1998-   Nonpatent document 27: The European Respiratory Journal,    Nagatsuka T. 2000, 16; 861-   Nonpatent document 28: The Journal of Infectious Diseases, McMurray    D N, 2000, 182: 861-   Nonpatent document 29: British Medical Journal, Kernoff P B A, 1977,    2: 1441-   Nonpatent document 30: The Journal of Immunology, Deniela S, 1989,    143: 1303-   Nonpatent document 31: Archives of Dermatological Research, Iverson    L, 1992, 284: 222-   Nonpatent document 32: Maki Hasegawa, 2005 Apr. 4, Allergic    inflammatory diseases—New approaches, Motohiro Kurosawa, 199-   Nonpatent document 33: Journal of the Oto-Rhino-Laryngological    Society of Japan (Nippon Jibiinkoka Gakkai Kaiho), Naoshi Yoshida,    2001, 104: 504-   Nonpatent document 34: Current Opinion in Allergy and Clinical    Immunology, Peter B, 2003, 3: 45-   Nonpatent document 35: Prostaglandins Leukotrienes and Essential    Fatty Acids, Gueck T. 2003, 68: 317-   Nonpatent document 36: Veterinary Dermatology, Gueck T. 2004, 15:    309-   Nonpatent document 37: Allergy, Shoso Yamamoto, 2004, 49: 45.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide an amount of DGLAsuitable for more safely and effectively prevent and treat atopicdermatitis.

We have found that PUFAs and their related metabolites becomequantitatively and qualitatively aberrant due to various factors, whichin turn causes undesirable biological changes, or conversely as a resultof undesirable biological changes, PUFAs and their related metabolitesoften become quantitatively and qualitatively aberrant, and under theseconditions, the basic principle is to correct the aberrant states bypositively ingesting PUFAs, and in particular, the n6-series PUFAs amongthe PUFAs are extremely useful, and work well at doses smaller than GLA,and thereby have completed the present invention.

Thus, the present invention provides a composition which comprises DGLAand which has the effect of preventing or treating skin diseases.

The composition is, for example, a food composition or a pharmaceuticalcomposition.

Dermatitis is, for example, allergic dermatitis or atopic dermatitis.

The content of DGLA in the composition is, for example, an ingestableamount of 5 mg-600 mg of DGLA per adult per day. For example, the amountof DGLA can be ingested at 5 mg-200 mg or 5 mg-150 mg of DGLA per adultper day.

DGLA may be present in the form of a glyceride, a phospholipid, aglycolipid, an alkylester, or a free fatty acid. The glyceride may be,for example, a triglyceride, a diglyceride, or a monoglyceride.Preferably said glyceride is a triglyceride and/or a diglyceride.

The composition may be in the form of, for example, a pill, a tablet ora capsule.

The composition of the present invention comprises DGLA and can take aform of a food or beverage indicated for the effect of preventing ortreating skin diseases. For example, it can be a food or beverageindicated for the effect of preventing or treating skin diseases, saidfood or beverage comprising an amount suitable to ingest 5 mg-600 mg ofDGLA per adult per day. Furthermore, it can be a food or beverageindicated for the effect of preventing or treating skin diseases, saidfood or beverage comprising an amount suitable to ingest 5 mg-200 mg ofDGLA per adult per day, or a food or beverage indicated for the effectof preventing or treating skin diseases said food or beverage comprisingan amount suitable to ingest 5 mg-150 mg of DGLA per adult per day.

It is another object of the present invention to provide a food orpharmaceutical composition that is safer and more effective for theprevention or treatment of various diseases closely related toeosinophil infiltration and increased cell count.

After intensive and extensive research in order to resolve the aboveproblems, the present inventors have found that dihomo-γ-linolenic acid(DGLA) is very useful in inhibiting eosinophil infiltration, and canmore effectively inhibit than other PUFAs, and thus have completed thepresent invention.

The present invention provides a composition which comprises DGLA andwhich has the effect of preventing or treating various diseases closelyrelated to eosinophil infiltration and increased cell count.

The composition is a food composition or a pharmaceutical composition.

The above diseases are skin diseases, such as atopic dermatitis, eczemaand psoriasis, respiratory system diseases, such as bronchial asthma,chronic obstructive pulmonary disease (COPD), hypersensitivitypneumonitis and eosinophilic pneumonia, and digestive system diseases,such as eosinophilic gastroenteritis and ulcerative colitis.

EPA and DHA are termed as the n-3 series PUFA as shown in FIG. 2,whereas DGLA belongs to the n-6 series PUFA based on the syntheticpathway in the body. DGLA is a highly safe food ingredient found inmeat, eggs, seafoods etc., but the content has been found to be muchlower than EPA or DHA or arachidonic acid of the same n-6 series PUFAs.As shown in U.S. Pat. No. 3,354,581, the present inventors have inventeda fermentation method for producing DGLA lipids by a microbial strain,thus enabling mass production of a triglyceride SUNTGD in which about40% of the constituent fatty acids are DGLA. Structurally speaking, DGLAhas three unsaturated bonds which are smaller than EPA or DHA, and thisingredient is refractory to oxidation thus emitting no orders and havingexcellent stability.

Regarding the physiological function of this ingredient, part of it hasbeen confirmed in vivo or in vitro: in vivo, effects of inhibitingplatelet agglutination (British Medical Journal, Kernoff P B A, 1977, 2:1441), delayed-type footpad edema (Lipids, Taki H., 1993, 28: 873),blood pressure increase (Lipids, Cedric H., 1984, 19: 699) and othereffects have been reported, and in vitro, effects of inhibiting theproduction of cytokines, such as interleukin-2, -10 and tumor necrosisfactor (TNF-α) (Immunology, Maaike M B W D, 2003, 110: 348; The Journalof Immunology, Deniela S, 1989, 143: 1303), leukotriene production(Archives of Dermatological Research, Iversen L, 1992, 284: 222), and Tcell growth (Prostaglandin Leukotrienes and Essential Fatty Acids,Zurier R B, 1999, 60: 371) and other effects have been reported.

However, no direct demonstration of the effect on eosinophils has beenmade. As described above, though DGLA is expected to be safe and bestabile, nothing is known on the physiological function thereof,specifically whether it has any effect on eosinophils, or the intensityof the effect, if any, compared to PUFAs.

DGLA may be present in the form of a glyceride, a phospholipid, aglycolipid, an alkylester, or a free fatty acid. Said glyceride may be,for example, a triglyceride, a diglyceride, or a monoglyceride.Preferably said glyceride is a triglyceride and/or a diglyceride.

The composition may be in the form of, for example, a pill, a tablet ora capsule.

The composition of the present invention comprises DGLA and can beincluded in a food or beverage for preventing or treating variousdiseases that are closely related to eosinophil infiltration andincreased cell count. For example, it can be a food or beverage forpreventing or treating various diseases that are closely related toeosinophil infiltration and increased cell count.

It is a further object of the present invention to provide a safer andmore effective food or a pharmaceutical composition for various diseasesthat are closely related to increased mast cell count.

After intensive and extensive research in order to resolve the aboveproblems, the present inventors have found that dihomo-γ-linolenic acid(DGLA) is very useful in inhibiting enhancement in a number of mastcells, and thus have completed the present invention.

Considering medically or nutritionally useful food ingredients, therecan be mentioned dihomo-γ-linolenic acid (DGLA), a n-6 seriespolyunsaturated fatty acid (PUFA) as shown in FIG. 2, as one of thecandidate materials. DGLA is abundantly found in meat, eggs, seafood andthe like, which have been ingested by mankind for a long period of timeand are naturally very safe. However, the content has been found to bemuch lower than arachidonic acid of the same n-6 series PUFAs or EPA orDHA. As shown in U.S. Pat. No. 3,354,581, the present inventors haveinvented a fermentation method for producing DGLA lipids by a microbialstrain, thus enabling mass production of a triglyceride SUNTGD in whichabout 40% of the constituent fatty acids are DGLA.

Regarding the physiological function of this ingredient, part of it hasbeen confirmed in vivo or in vitro: in vivo, effects of inhibitingplatelet agglutination (British Medical Journal, Kernoff P B A, 1977, 2:1441), delayed-type footpad edema (Lipids, Taki H., 1993, 28: 873),blood pressure increase (Lipids, Cedric H., 1984, 19: 699) and othereffects have been reported, and in vitro, effects of inhibiting theproduction of cytokines such as interleukin-2, -10 and tumor necrosisfactor (TNF-α) (Immunology, Maaike M B W D, 2003, 110: 348; The Journalof Immunology, Deniela S, 1989, 143: 1303), leukotriene production(Archives of Dermatological Research, Iversen L, 1992, 284: 222), and Tcell growth (Prostaglandin Leukotrienes and Essential Fatty Acids,Zurier R B, 1999, 60: 371) and other effects have been reported.However, no direct demonstration of the effect on eosinophils has beenmade.

On the other hand, linoleic acid (LA) and γ-linolenic acid (GLA) amongthe same n-6 series PUFAs, effects on mast cells have been investigated,and the former has been reported to enhance the release ofstimulation-responsive histamine from lined mast cells (ProstaglandinLeukotrienes and Essential Fatty Acids, Gueck T. 2003, 68: 317). Thelatter has been found to inhibit histamine release in the same testsystem (Veterinary Dermatology, Gueck T. 2004, 15: 309). However, bothstudies examined the effect on the release of chemical mediators frommast cells, however, their effect on the number of mast cells is notknown.

As described above, it was not known what effects DGLA might have on thenumber of mast cells.

Thus, the present invention provides a composition which comprises DGLAand which prevents or treats various diseases closely related toincreased cell count.

The composition is a food composition or a pharmaceutical composition.

The above diseases are skin diseases, such as atopic dermatitis, eczemaand psoriasis, basal cell carcinoma, and prickle cells, bronchialasthma, pollinosis, allergic rhinitis, allergic conjunctivitis and thelike.

DGLA may be present in the form of a glyceride, a phospholipid, aglycolipid, an alkylester, or a free fatty acid. The glyceride may be,for example, a triglyceride, a diglyceride, or a monoglyceride.Preferably the glyceride is a triglyceride and/or a diglyceride.

The composition may be in the form of, for example, a pill, a tablet ora capsule.

The composition of the present invention comprises DGLA and can take aform of a food or beverage indicated for the effect of preventing ortreating various diseases that are closely related to increased mastcell counts. For example, it can be a food or beverage indicated for theeffect of preventing or treating various diseases that are closelyrelated to increased mast cell counts.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a drawing showing the metabolic pathway of n-6 seriespolyunsaturated fatty acids (PUFAs).

FIG. 2 shows the metabolic pathways of n-6 series and n-3 seriespolyunsaturated fatty acids.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be explained in detail below.

As food compositions, there can be mentioned food supplements and(medical) prescribed products and preparations such as tablets, pillsand capsules. Furthermore, there can be mentioned solid or liquidfoodstuffs, for example dairy products (margarine, butter, milk,yogurt), bread, cake; drinks, for example beverages (tea, coffee, cocoa,chocolate drinks), fruit juice, soft drinks (for example carbonateddrinks); confectioneries; oily foods (snacks, salad dressings,mayonnaise), soup, sauce, carbohydrate-rich foods (rice, noodles,pasta), fish-containing foods, baby food (for example, baby formula, asa liquid or a powder), pet food, and prepared foods or microwavablefood.

DGLA may be derived from any suitable source. However, there are fewknown natural lipid sources having a high DGLA content, and minuteamounts may be extracted from cow's liver, a pig's kidney, egg-yolk,etc. With the progress in the microbial fermentation technology inrecent years, it may be derived from microorganisms, for example fungi,bacteria or yeast.

Suitable fungi belong to the order of Mucorales, for exampleMortierella, Pythium or Entomophyhora. A suitable source of DGLA isMortierella. More preferably it is derived from Mortierella alpina. As aDGLA-containing lipid, a triglyceride in which about 40% of theconstituent fatty acids are DGLA may be prepared by a microbialfermentation method using Mortierella.

In addition to DGLA, one or more additive PUFAs may be supplied. Inaddition to DGLA, it may be another n-6 series PUFA (for examplelinoleic acid (LA)), γ-linolenic acid (GLA) and arachidonic acid (AA))or a n-3 series PUFA (for example EPA, DHA).

As a physiologically acceptable derivative of this acid that can beconverted to DGLA for use in the present invention, there can bementioned the form of a DGLA-containing triglyceride, diglyceride, andmonoglyceride, or a phospholipid, a glycolipid, a free fatty acid, afatty acid ester (for example, methyl or ethyl ester), and a sterolester.

Preferably, PUPA is present in an oil. This can be a pure oil, aprocessed oil (for example, a chemically and/or enzymatically processedoil), or a concentrated oil. Though these oils may contain 10-100% ofPUFAs, the content of the desired PUFA, for example DGLA, may be 5% ormore if the oil is derived from a microorganism, preferably 10% or more,more preferably 25% or more. The oil may contain one or more PUFAswithin the concentration range of the above percentage. The oil may be asingle oil derived from a single cell or a microorganism, or may be ablended or mixed oil of two or more oils derived from different sources.The oil may contain one or more additives, for example an antioxidant(for example, tocopherol, vitamin E, tocotrienol, an ascorbic acidderivative, a palmitate or an ester, astaxanthin), sesamine, CoQ10 orthe like.

The present invention may be used to enhance the PUFA level of a normalhealthy individual who has had a sufficient meal or an individual whoshows a normal PUFA level for the purpose of preventing diseases, andmaintaining health.

However, it can also be used for individuals whose PUFA levels are lowor insufficient. For example, it can be used for prophylaxis,prevention, improvement and treatment of diseases or conditions relatedto abnormal or low levels of n-3 series or n-6 series PUFAs in blood.Thus, the present invention provides application in subjects who havelow DGLA levels, for example subjects who cannot convert and/oreffectively convert LA to GLA and DGLA or GLA to DGLA. Thus, inappropriate patients, Δ6-desaturase and/or carbon chain elongase may bedysfunctional, insufficient or deficient.

The present invention specifically provides the use in humans having lowlevels of DGLA; for example when an immunological level of atopicdermatitis, is low, immunological level is decreased or enhanced, andthe present invention provides the use to such subjects at an abnormalimmunological state.

It can also be used to correct states in which DGLA levels are low orDGLA levels are not normal, and other states, for example various skindiseases such as dermatitis, eczema and UV-derived skin disorders,rheumatoid arthritis, diabetes mellitus, alcoholics and smokers.

Specifically the present invention provides the use in subjects withvarious diseases closely related to eosinophil infiltration andincreased cell count, such as skin diseases, respiratory system diseasesand digestive system diseases.

Skin diseases may indicate atopic dermatitis, eczema and psoriasis,respiratory system diseases may indicate bronchial asthma, chronicobstructive pulmonary disease (COPD), hypersensitivity pneumonitis andeosinophilic pneumonia, and digestive system diseases may indicateeosinophilic gastroenteritis and ulcerative colitis.

Specifically the present invention provides the use in subjects withvarious diseases in which increased mast cell count has been observed,and may indicate skin diseases, such as atopic dermatitis, eczema,psoriasis, basal cell carcinoma and prickle cell carcinoma, bronchialasthma, pollinosis, allergic rhinitis, allergic conjunctivitis and thelike.

EXAMPLES

The present invention will now be explained more specifically below.

Example 1

Since it was believed that ingestion of DGLA is useful for theprevention or treatment of atopic dermatitis, as described above, itsusefulness in experimental animals was investigated using a triglycerideSUNTGD, a DGLA lipid having DGLA as the main constituent fatty acid,that was prepared according to the method described in U.S. Pat. No.3,354,581. As an animal model of atopic dermatitis, NC/Nga mice wereused in this study. Since mice have been recognized as one of the mostuseful models of atopic dermatitis at present, and steroid externalpreparations and immunosuppressive external preparations currently usedin the clinical setting for the treatment of atopic dermatitis have alsobeen demonstrated to be effective in mice it has widely been used inscreening of therapeutic agents for atopic dermatitis.

In a conventional feeding environment, mice are known to spontaneouslydevelop dermatitis at about 8 weeks after birth, and then theinflammation aggravates to a chronic type with the passage of days, anddevelop human atopic dermatitis-like symptoms both macroscopically andhistopathologically. As characteristics of this pathological condition,there can be mentioned increased serum IgE accompanied by the onset ofdermatitis and marked infiltration of immunocompetant cells, such asmast cells, eosinophils and T cells at the lesion.

In this study, male or female NC/Nga mice were prepared and three groupsof seven mice per group were used in a conventional feeding environment.The following three types of diets shown in FIG. 1 were prepared, andwere fed ad libitum at post-ablactation week 5 and until the completionof the study at week 12. The groups comprised a control diet group, ahigh DGLA diet group and a low DGLA diet group SUNTGD was added to thediet of the latter two groups, at about 1.0% DGLA (calculated as theamount of free fatty acids), for the high DGLA group, and at about 0.5%DGLA (calculated as the amount of free fatty acids) for the low DGLAgroup. Since the mean body weight of the mice was 20 g and the meandaily diet ingestion was about 2 g, the amount ingested of DGLA in thisexperiment was estimated to be about 1000 mg/kg per day for the highDGLA group, and about 500 mg/kg per day for the low DGLA group. Theamount of total fatty acids in the diet was adjusted to be 5% for all ofthe groups. The items evaluated were the macroscopic score of dermatitissymptoms under a blinded condition, scratching behavior and plasma IgE.

As a result, without exhibiting any aberration in body weight both ofthe DGLA diet groups exhibited a statistically significant inhibitoryeffect in all of the evaluation items of the macroscopic score ofdermatitis symptoms (Table 2), scratching behavior (Table 3), amount ofplasma IgE produced (Table 4), suggesting the possibility that theingestion of DGLA may be useful for the prevention of atopy. The fattyacid composition (Table 5) in the plasma at this time reflected theeffect of the diet, and in all of the organs, increases in the amount ofDGLA and decreases in the amount of LA were noted, dependent on the doseof the DGLA diet, with the variation in fatty acid composition beingmost conspicuous in the spleen, suggesting that DGLA may have a greatimpact on the physiological function of the immune system. Moresurprisingly, the preventive effect was independent of the dose of DGLA,i.e. the effect of preventing atopy tended to be more prominent in thelow DGLA diet group compared to the high DGLA diet group.

As described above, the fatty acid composition in the living bodyexhibited a dose dependent increase in DGLA, it is obviously not theattenuation of the effect by inhibiting the absorption of DGLA per seetc. This strongly suggests the possibility that there may be an optimumamount of DGLA for preventing atopy in mice or humans, i.e. an amountlower than that of the low DGLA diet used in this study may exhibit thehighest inhibitory effect. Furthermore, in the case of variation in theamount of DGLA in the plasma by the DGLA diet, DGLA in plasmaphospholipids was 4.3% in the low DGLA diet group, which wasspecifically effective in the improvement of atopic dermatitis-likesymptoms, and in the control diet group it was 1.0%, which indicatesabout 4-fold or 3% by weight increase revealing that such an enhancementin the amount of plasma DGLA is one of the parameters that mostcontribute to the therapeutic effect of atopic dermatitis.

If the ingestion of DGLA could promote such a correction in the amountof DGLA in humans, atopic dermatitis is expected to be improved. For themice model, as described above, GLA has been reported (Abstract of the50th General Meeting of the Japanese Society of Allergology, Zui Hamada,2000, pp. 999), and the dose therein was about 1250 mg/kg per day,whereas the amount of the low DGLA diet used was about 500 mg/kg per dayor less. From this fact as well, it may be expected that DGLA canprevent atopic dermatitis more efficiently than GLA. Furthermore, ALA, an-3 series PUFA, has been investigated in the same model (ProstaglandinLeukotrienes and Essential Fatty Acids, Suzuki R, 2002, 66: 43) wherein,although the exact dose is not known, no effect of preventing atopicdermatitis, such as the correction of dermatitis symptoms and blood IgEcan be recognized even though ALA in the erythrocyte membrane in theliving body becomes markedly increased in a diet containing a largeamount of ALA.

TABLE 1 Table of ingredients in each diet (unit: %) Control High DGLALow DGLA Ingredient (%) diet diet diet Casein 20.0 20.0 20.0DL-methionine 0.3 0.3 0.3 Cornstarch 45.0 45.0 45.0 Pregelatinized 10.010.0 10.0 cornstarch Sucrose 10.0 10.0 10.0 Cellulose powder 5.0 5.0 5.0AIN76 mineral mix 3.5 3.5 3.5 AIN76 vitamin 1.0 1.0 1.0 blend Choline0.2 0.2 0.2 bitartarate Corn oil 3.34 0.84 2.09 Lard oil 1.67 0.42 1.04SUNTGD(*1) 0.00 (*2)2.50 (*3)1.25 Olive oil 0.00 1.25 0.63 Total 100.0100.0 100.0 (*1)A triglyceride in which about 40% of the constituentfatty acids are DGLA (*2)Corresponds to about 1.0% in the diet as theamount of free DGLA (*3)Corresponds to about 0.5% in the diet as theamount of free DGLA

TABLE 2 Scores of dermatitis symptoms of NC/Nga mice (mean ± standarddeviation, N = 7) At 10-week-old At 12-week-old Control diet group  7.1± 0.5  9.1 ± 1.0 High DGLA diet group *2.1 ± 1.0 *3.0 ± 0.5 Low DGLAdiet group *1.4 ± 0.3 *1.6 ± 0.4 *p < 0.05 (Dunnett's test vs. thecontrol group)

TABLE 3 Scratching behavior in NC/Nga mice (the number of scratching per20 minutes, mean ± standard deviation, N = 7) At 10-week-old At12-week-old Control diet group  75.9 ± 10.7 51.7 ± 9.1 High DGLA dietgroup *18.0 ± 8.9 35.0 ± 7.1 Low DGLA diet group *21.4 ± 6.7 **21.9 ±5.7  *p < 0.05, **p < 0.01 (Student t-test vs. the control group)

TABLE 4 Total plasma IgE in 12-week-old NC/Nga mice (μg/ml, mean ±standard deviation, N = 7) At 12-week-old Control diet group 64.2 ± 39.8High DGLA diet group 29.8 ± 21.0 Low DGLA diet group **15.6 ± 3.6   *p <0.01 (Dunnett's test vs. the control group)

TABLE 5 Phospholipid fatty acid composition in the plasma of 12-week-oldNC/Nga mice (% of phospholipid in the total fatty acids, mean ± standarddeviation, N = 7) Fatty acid Control diet High DGLA diet Low DGLA dietLA 15.4 ± 4.1  **5.6 ± 2.0  **8.4 ± 2.6  20:0/18:3G 0.0 ± 0.1 0.1 ± 0.10.0 ± 0.0 DGLA 1.0 ± 0.4 **7.6 ± 4.0  **4.3 ± 1.5  AA 10.3 ± 2.0  **15.7± 3.7   **16.5 ± 1.6   EPA 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 DHA 2.2 ± 0.82.3 ± 0.4 1.8 ± 0.6 **p < 0.01 (Student t-test vs. the control group)

Example 2

In order to clarify the relationship between the amount of DGLA orallyingested in humans and the internal DGLA level, the amount of DGLAorally ingested was investigated by examination of the diet, and afterdrawing blood, the amount of DGLA in the serum phospholipids wasdetermined. The subjects were eleven healthy males aged 60-70, and wereasked to keep a diary of the content of their diets. From the diary, theamount ingested by each food material was determined for meat, eggs, andseafood known to contain DGLA, and using the DGLA content of each foodmaterial calculated from “Fourth revised STANDARD TABLES OF FOODCOMPOSITION IN JAPAN, Standard Tables of Dietary Lipids in Japan(1990)”, the amount ingested of DGLA per day per person was calculated.From the amount of DGLA ingested per week, the mean amount of DGLAingested per day was calculated for each person.

On the other hand, blood was drawn the day after the last entry of thediary, and for the serum phospholipid fractions obtained, the fatty acidcomposition was analyzed according to a standard method. Thus, from theserum the lipid component extracted by the Folch method, a standardmethod, and the lipid was fractionated on a thin layer chromatography(the developing solution is hexane/diethyl ether=7/3) to obtain thephospholipid fraction. The silica gel layer was directly scraped, andwas reacted in hydrochloric acid-methanol at 50° C. for 3 hours,followed by extraction with hexane to obtain a fatty acid methyl estermixture. As the internal standard, pentadecanoic acid was used. Thefatty acid methyl ester mixture was analyzed by a capillary gaschromatography to obtain DGLA (% by weight) in the total fatty acids inthe serum phospholipid.

The result demonstrated that there is a positive correlation between themean daily amount ingested of DGLA and DGLA (% by weight) in the serumphospholipid. When X=the mean daily amount (mg) ingested of DGLA andY=DGLA (% by weight) in the serum phospholipid, the correlation linearequation becomes Y=0.0312 X+1.361, indicating a relationship that witheach increase of about 32 mg in the amount of DGLA ingested, DGLA in theserum phospholipid increases by 1% by weight. Further known from thisresult is the tendency that the changes in blood DGLA after oralingestion of DGLA are more pronounced in humans than in mice. In theearlier result on mice, it was established that, for the low DGLA dietgroup, by ingesting 500 mg/kg of DGLA in terms of body weight forsubstantially 8 weeks, the amount of DGLA in the plasma phospholipidincreases 3% by weight.

In the study on humans, when the body weight is 60 kg, by calculatingthat by ingestion of 32 mg/60 kg=0.53 mg/kg of DGLA, the amount of DGLAin the serum phospholipid increases 1% by weight. Also, when DGLA in theplasma phospholipid increased about 3% by weight in mice, the symptomsof atopic dermatitis were effectively improved, and conversely inhumans, the amount of DGLA required to increase the amount of DGLA inserum phospholipid about 3% by weight, may be about 100 mg per day.Further the amount of orally ingested GLA that can improve atopicdermatitis in humans is about 180-1440 mg per day, and is stronglysuggested that smaller amounts of DGLA can improve atopy.

Example 3

For some skin diseases, it is known that the skin may be damaged bysunlight. Specifically, ultraviolet light classified as UV-B atwavelengths of 290-320 nm is considered a major factor responsible foracute sunburn of the skin, pigmentation, suntan, skin cancer and thelike. In order to explore the further possibility of DGLA affecting skinfunction, the effect of DGLA on acute inflammation of the skin wasevaluated using an UV-B-induced acute inflammation guinea pig model.This model animal is one model widely used for drug screening in thedevelopment of pharmaceutical agents intended for anti-inflammatoryeffects.

In this study, male Hartley guinea pigs were prepared and divided into acontrol diet group and a DGLA diet group. Each diet group was continuedto be given ad libitum for three weeks, from 5 week-old to 7 week-oldanimals, at the completion of the experiment. The DGLA diet used thistime is different from the composition of the mouse diet describedearlier and was adjusted to about 0.08% of DGLA (calculated as the freefatty acid) in the diet. Thus, since the mean body weight of the guineapigs was about 400 g and the mean daily amount ingested was about 30 g,the amount ingested of DGLA in this experiment was estimated to be about60 mg/kg per day. After three weeks of free access to the diet, the backof the guinea pig was shaved, and after fixing the animals on anabdominal position, they were irradiated using a UV irradiator(Dermaray™, type M-DMR-I, Eisai) and a UV-B tube (FL-20S-E-30 lamp,central wavelength 305 nm, Toshiba) at a distance of 5 cm from the tubefor 15 minutes to induce an acute inflammatory reaction. After theinduction of skin erythema reaction, it was scored under a blindcondition according to the Draize method.

The result as shown in Table 6 indicated that the DGLA diet groupexhibited a tendency to inhibit an acute skin inflammatory reactionresulting from UV-B, specifically a statistically significant inhibitoryeffect at one hour after exposure to UV-B. At this time it was confirmed(though the result is not shown) that the amount of DGLA in thephospholipid of the plasma, the skin and other organs significantlyincreases in the DGLA diet group compared to the control diet group.Based on the above result, it was found that a DGLA diet is useful inthe prevention of UV-induced skin damage.

TABLE 6 Skin erythema score in UV-B-induced acute inflammation guineapig model (% in the total fatty acids, mean ± standard deviation) Timeelapsed after Control diet # DGLA diet exposure to UV-B (N = 12) (N =13)  1 hour later 1.8 ± 0.5 *1.3 ± 0.4   3 hours later 2.2 ± 0.5 1.9 ±0.6 24 hours later 1.7 ± 0.8 1.4 ± 0.7 48 hours later 1.5 ± 0.8 1.2 ±0.8 *p < 0.05 (Mann-Whitney U test vs. the control diet group) #: ThisDGLA diet contained the amount of free DGLA corresponding to about 0.08%in the diet.

Example 4

In order to investigate whether the ingestion of DGLA may have anyeffect on eosinophils, a triglyceride SUNTGD, a DGLA lipid having DGLAas the main constituent fatty acid, that was prepared according to amethod described in U.S. Pat. No. 335,458 was used to examine itsusefulness in experimental animals. This time, as an animal model thatis closely related to eosinophil infiltration and increased cell counts,NC/Nga mice were used. Since this animal model has been recognized to beone of the most useful models of atopic dermatitis at present, andsteroid external preparations and immunosuppressive externalpreparations currently used in the clinical setting for the treatment ofatopic dermatitis have been demonstrated to be effective in this animalmodel too, it has been widely used in screening of therapeutic agentsfor atopic dermatitis.

Under a conventional feeding environment, mice are known tospontaneously develop dermatitis at about 8 weeks after birth, and theninflammation aggravates to a chronic type with the passage of days, anddevelops a human atopic dermatitis-like symptom both macroscopically andhistopathologically. As characteristics of this pathological condition,there can be mentioned increased serum IgE accompanied by the onset ofdermatitis and marked infiltration of immunocompetant cells such as mastcells, eosinophils and T cells at the lesion.

In this study, male or female NC/Nga mice were prepared and two groupsof seven animals per group were used in a conventional feedingenvironment. The following two types of diets shown in Table 7 wereprepared, and were fed ad libitum at post-ablactation week 5, and untilthe completion of the study at week 12. The groups comprised a controldiet group and a DGLA diet group, and SUNTGD, a triglyceride having DGLAas the main constituent fatty acid, was added to the latter groups dietat about 1.0% DGLA (calculated as the amount of free fatty acids). Sincethe mean body weight of the mice was 20 g and the mean daily dietingestion was about 2 g, the amount ingested of DGLA in this experimentwas estimated to be about 1000 mg/kg per day for the DGLA group. Also,the amount of total fatty acids in the diet was adjusted to be 5% inboth groups. Items evaluated were the macroscopic score of dermatitissymptoms under a blinded condition, the number of times of scratchingsand IgE in the blood.

The animals were dissected upon completion of the experiment at week 12to remove the skin at the back of the neck which was a dermatitislesion, and then the tissue was fixed in a neutral buffered formalinsolution and embedded in paraffin to prepare sliced sections, which weresubjected to Luna stain to identify eosinophils. From these tissuesamples, three samples out of seven per group were selected, and for atotal of six samples, the degree of eosinophil infiltration wasrelatively evaluated. The method of selecting samples showed threesamples that exhibited a value close to the mean macroscopic score ofthe dermatitis symptoms of each group were selected. The relativeevaluation was made under a blinded condition so that the rater cannotidentify the sample he/she is evaluating. The evaluation criteria of thedegree of eosinophil infiltration was as follows:

++: Specifically severe eosinophil infiltration is confirmed at severalspots and infiltration is also severe as a whole;

+−: Eosinophil infiltration is confirmed at a few spots or almostnothing.

As a result, without exhibiting any aberration in body weight changes(Table 8) and general findings, the DGLA diet group exhibited a tendencyto suppress the number of eosinophils infiltrating into the skin lesioncompared to the control diet group as shown in Table 9. Furthermore, atthis time, alleviation in the macroscopic score of the dermatitissymptoms (the control diet group: 9.1±1.0, the DGLA diet group:3.0±0.5), suppression of the number of times of scratching (the controldiet group: 51.7±9.1 times, the DGLA diet group: 35.0±7.1 times),suppression of the amount of plasma IgE produced (the control dietgroup: 64.2±39.8 μg/ml, the DGLA diet group: 29.8±21.0 μg/ml) were alsoconfirmed. The above results suggest that ingestion of DGLA may be veryuseful for various diseases that are closely related to eosinophilinfiltration and increased cell count.

TABLE 7 Table of ingredients in each diet (unit: %) Ingredient (%)Control diet DGLA diet Casein 20.0 20.0 DL-methionine 0.3 0.3 Cornstarch45.0 45.0 Pregelatinized 10.0 10.0 cornstarch Sucrose 10.0 10.0Cellulose powder 5.0 5.0 AIN76 mineral mix 3.5 3.5 AIN76 vitamin 1.0 1.0blend Choline 0.2 0.2 bitartarate Corn oil 3.34 0.84 Lard oil 1.67 0.42SUNTGD(*1) 0.00 (*2)2.50 Olive oil 0.00 1.25 Total 100.0 100.0 (*1)Atriglyceride in which about 40% of the constituent fatty acids is DGLA.(*2)Corresponding to about 1.0% in the diet as the amount of free DGLA.

TABLE 8 Changes in body weight of NC/Nga mice (g, mean ± standarddeviation, N = 7) Control diet DGLA diet Week-old group group 6 week-old20.4 ± 3.7 20.0 ± 2.4 9 week-old 21.9 ± 3.1 22.9 ± 2.5 12 week-old  23.2± 3.8 25.4 ± 3.2

TABLE 9 Degree of eosinophil infiltration in the skin at the back of theneck of NC/Nga mice Degree of eosinophil Sample I.D. infiltrationControl diet Sample 1 ++ group Sample 2 ++ Sample 3 ++ DGLA diet groupSample 4 +− Sample 5 +− Sample 6 +− Evaluation criteria of the degree ofeosinophil infiltration: ++: Specifically severe eosinophil infiltrationis confirmed at several spots and infiltration is also severe as awhole; +−: Eosinophil infiltration is confirmed at a few spots or almostnothing.

As described above, eosinophil infiltration can be inhibited by DGLA.

Example 5

In order to investigate whether ingestion of DGLA may have any effect onincreased mast cell count, a triglyceride SUNTGD, a DGLA lipid havingDGLA as the main constituent fatty acid, that was prepared according toa method described in U.S. Pat. No. 335,458, was used to examine itsusefulness in experimental animals. This time, as an animal model thatis closely related to increased mast cell count, NC/Nga mice were used.Since this animal model has been recognized to be one of the most usefulmodels of atopic dermatitis at present, and steroid externalpreparations and immunosuppressive external preparations currently usedin the clinical setting for the treatment of atopy have also beendemonstrated to be effective in this animal model, it has been widelyused in screening of therapeutic agents for atopic dermatitis.

Under a conventional feeding environment, mice are known tospontaneously develop dermatitis at about 8 weeks after birth, and theninflammation aggravates to a chronic type with the passage of days, anddevelops a human atopic dermatitis-like symptom both macroscopically andhistopathologically. As characteristics of this pathological condition,there can be mentioned increased serum IgE accompanied by the onset ofdermatitis, differentiation and proliferation of mast cells at thelesion, and marked infiltration of immunocompetant cells, such aseosinophils and T cells at the lesion.

In this study, male or female NC/Nga mice were prepared and two groupsof seven animals per group were used in a conventional feedingenvironment. The following two types of diets shown in Table 10 wereprepared, and were fed ad libitum at post-ablactation week 5, and untilthe completion of the study at week 12. The groups comprised a controldiet group and a DGLA diet group, and SUNTGD, a triglyceride having DGLAas the main constituent fatty acid, was added to the latter groups dietand adjusted to be about 1.0% DGLA (calculated as the amount of freefatty acids). Since the mean body weight of the mice was 20 g and themean daily diet ingestion was about 2 g, the amount ingested of DGLA inthis experiment was estimated to be about 1000 mg/kg per day for theDGLA group. Also, the amount of total fatty acids in the diet wasadjusted to be 5% in both groups. Items evaluated were the macroscopicscore of dermatitis symptoms under a blinded condition, the number oftimes of scratchings, and IgE in the blood.

The animals were dissected upon completion of the experiment at week 12to remove the skin at the back of the neck, which was a dermatitislesion, and then the tissue was fixed in a neutral buffered formalinsolution, and embedded in paraffin to prepare sliced sections, whichwere subjected to toluidine blue stain to identify mast cells. Fromthese tissue samples, two samples out of seven per group were selected,and for a total of four samples, the mast cell count and the degree ofthe cell count was evaluated. The method of selecting samples was thattwo samples that exhibited a value close to the mean macroscopic scoreof the dermatitis symptoms of each group were selected. In order tocount the number of mast cells, the number of mast cells confirmed undermicroscopic examination at 10×40 fold magnification were counted, andthe counting was repeated five times at different fields for the samesample, and the mean thereof was designated as the mast cell count.

For the relative evaluation of mast cell count, the mast cell count inthe sample as a whole was evaluated. The relative evaluation criteriawas as follows:

++: Extremely abundant mast cells are confirmed at several spots, andabundant as a whole;

+: Abundant mast cells are confirmed at a few spots, and slightlyabundant as a whole;

+−: Mast cells are at an approximately normal level.

Counting of the number of mast cells and the relative evaluation of thecell count were made under a blinded condition so that the rater cannotidentify the sample he/she is evaluating.

As a result, without exhibiting any aberration in body weight changes(Table 11) and general findings, the DGLA diet group exhibited atendency to suppress the number of mast cells in the skin lesioncompared to the control diet group as shown in Table 12. Furthermore, atthis time, alleviation in the macroscopic score of the dermatitissymptoms (the control diet group: 9.1±1.0, the DGLA diet group:3.0±0.5), suppression of the number of times of scratching (the controldiet group: 51.7±9.1 times, the DGLA diet group: 35.0±7.1 times),suppression of the amount of plasma IgE produced (the control dietgroup: 64.2±39.8 μg/ml, the DGLA diet group: 29.8±21.0 μg/ml) were alsoconfirmed. The above result suggests that the ingestion of DGLA may bevery useful for various diseases that are closely related to increasedmast cell counts such as atopic dermatitis, bronchial asthma, andallergic rhinitis.

TABLE 10 Table of ingredients in each diet (unit: %) Ingredient (%)Control diet DGLA diet Casein 20.0 20.0 DL-methionine 0.3 0.3 Cornstarch45.0 45.0 Pregelatinized 10.0 10.0 cornstarch Sucrose 10.0 10.0Cellulose powder 5.0 5.0 AIN76 mineral mix 3.5 3.5 AIN76 vitamin 1.0 1.0blend Choline 0.2 0.2 bitartarate Corn oil 3.34 0.84 Lard oil 1.67 0.42SUNTGD(*1) 0.00 (*2)2.50 Olive oil 0.00 1.25 Total 100.0 100.0 (*1)Atriglyceride in which about 40% of the constituent fatty acids are DGLA.(*2)Corresponding to about 1.0% in the diet as the amount of free DGLA.

TABLE 11 Changes in body weight of NC/Nga mice (g, mean ± standarddeviation, N = 7) Control diet DGLA diet Week-old group group 6 week-old20.4 ± 3.7 20.0 ± 2.4 9 week-old 21.9 ± 3.1 22.9 ± 2.5 12 week-old  23.2± 3.8 25.4 ± 3.2

TABLE 12 Number of mast cells in the skin at the back of the neck ofNC/Nga mice and relative evaluation Mast cell Relative Sample I.D. count(*1) evaluation (*2) Control diet Sample 1 33.2 ++ group Sample 2 37.4++ DGLA diet Sample 3 17.4 + group Sample 4 13.0 +− (*1) Mast cellcount: The number of mast cells confirmed in a field at 10 × 40 foldmagnification. The counting was repeated five times at different fieldsfor the same sample, and the mean thereof is expressed. (*2) Relativeevaluation criteria for mast cell count: ++: Extremely abundant mastcells are confirmed at several spots, and abundant as a whole; +:Abundant mast cells are confirmed at a few spots, and slightly abundantas a whole; +−: Mast cells are at an approximately normal level.

INDUSTRIAL APPLICABILITY

In the examination of the effect of DGLA ingestion for atopicdermatitis, DGLA can prevent dermatitis at lower doses than GLA, and isthus more useful in food for preventing atopic dermatitis. Furthermore,ingestion at the most appropriate dose is extremely important forusefulness.

Since the effect of oral ingestion of DGLA for inhibiting eosinophilinfiltration is more effective than other PUFAs, DGLA is more useful invarious diseases closely related to eosinophil infiltration andincreased cell count, such as skin diseases, respiratory system diseasesand digestive system diseases compared to other PUFAs.

Since oral ingestion of DGLA can suppress increased mast cell count verysafely and effectively, DGLA is highly useful in various diseasesclosely related to increased mast cell count, such as skin diseases,asthma and rhinitis.

1-40. (canceled)
 41. A method for treating skin diseases in anindividual, consisting essentially of administering dihomo-γ-linolenicacid (DGLA) to the individual in an amount sufficient to treat skindiseases in the individual, wherein said DGLA is derived from amicroorganism, and wherein DGLA is administered at an amount of 5 mg-600mg per day.
 42. The method of claim 41, wherein the skin disease isatopic dermatitis.
 43. The method of claim 41, wherein the individual isan adult.
 44. The method of claim 41, wherein the DGLA is in a food orbeverage, or a pharmaceutical composition.
 45. The method of claim 44,wherein said pharmaceutical composition is in the form of a solution, apill, a tablet or a capsule.
 46. The method of claim 41, wherein saiddihomo-γ-linolenic acid (DGLA) is in the form of a glyceride, aphospholipid, a glycolipid, an alkylester, or a free fatty acid or asalt thereof.
 47. The method of claim 46, wherein said glyceride is atriglyceride, diglyceride, or a monoglyceride.
 48. The method of claim41, wherein the individual has as low dihomo-γ-linolenic acid (DGLA)level.
 49. The method of claim 48, wherein the individual having a lowdihomo-γ-linolenic acid (DGLA) level has dysfunctional Δ6 desaturaseand/or carbon chain elongase, has insufficient Δ6 desaturase and/orcarbon chain elongase, or lacks Δ6 desaturase and/or carbon chainelongase.
 50. The method of claim 44, wherein the food or beverage isbaby food.
 51. The method of claim 41, wherein dihomo-γ-linolenic acid(DGLA) is administered at an amount of 0.08 mg/kg-3.3 mg/kg body weightof said individual per day.
 52. The method of claim 41, whereindihomo-γ-linolenic acid (DGLA) is administered at an amount of 0.08mg/kg-2.5 mg/kg body weight of said individual per day.
 53. The methodof claim 41, wherein dihomo-γ-linolenic acid (DGLA) is administered atan amount of 5 mg-200 mg per day.
 54. The method of claim 41, whereindihomo-γ-linolenic acid (DGLA) is administered at an amount of 5 mg-150mg per day.